Cloning and characterization of ovine alphaS1-casein gene promoter: a transfection study in rat mammary gland cell line.

Promoter regions of milk protein genes are frequently used to produce pharmaceutically and medically important proteins in the mammary gland of transgenic animals and also can be used for the construction of an inducible eukaryotic expression vector. The aim of the present study was to clone, sequence and characterize the regulatory elements in ovine alphaS1-CSNGP. For the first time we have cloned and sequenced region extending from - 2136 to +49 bp containing 5'-flanking region and exon I. Computational analysis of the sequence showed presence of core promoter elements viz., TATA box, CAAT box and initiator sequence. Mammary gland specific sequences included MGF/STAT 5, MPBF, Yu Lee 2, 4 and 5, Oka box C and hormone responsive elements (HRE) viz., GRE, PRE, PRL, IRE and also Polyoma enhancer 3 sequences. Computational analysis data is validated by following the reporter gene expression studies in rat breast cell line. Six reporter gene constructs under the control of full length, proximal, distal, minimal and proximal-distal fused promoter segments were constructed to assess the effect of presence or absence of few selected regulatory elements on expression ability of the promoter. Based on qualitative evaluation of fluorescence, the pGFP-F/VspI showed highest fluorescence followed by pGFP-P, pGFP-F/SpeI, pGFPminimal and pGFP-D.     

人类连接蛋白基因Cx26的上游调控部位

人类连接蛋白２６（Ｃｏｎｎｅｘｉｎ ２６,Ｃｘ２６）已被看作乳腺癌上皮细胞中的抑瘤基因候选者．为了阐明此基因的调控机理,对其转译起始点上游的一个具有启动功能的１．６ ｋｂ 片段采用ｅｘｏ Ⅲ构建１０个单向删除重组体后进行了ＣＡＴ报告基因分析．结果表明,此１．６ ｋｂ 片段其启动子功能部位位于５′端２００ ｂｐ 范围内．其中含有－ＴＧＴ盒（位于１８２～１８７ ｂｐ）,一个ＴＴＡＡＡＡ 盒位于１５８～１６３ ｂｐ,这是人类Ｃｘ２６基因的又一启动区,这些发现无疑地对了解和阐明Ｃｘ２６基因在乳腺发育过程中及其病理生理作用的复杂调控具有特别重要的意义．     

Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human ε-Globin Gene

The human β-like globin genes are arranged as a clusterof five genes (ε, Gγ, Aγ, δ and β) in the order of theirtemporal expression. The human embryonic ε-globin geneis expressed in the blood island of the embryonic yolk sacand is silenced completel     

Analysis of rat prolactin promoter sequences that mediate pituitary-specific and 3',5'-cyclic adenosine monophosphate-regulated gene expression in vivo

To determine the rat PRL (rPRL) promoter sequences that mediate pituitary-specific and cAMP-induced gene expression in vivo, various lengths of the rPRL promoter were ligated to the luciferase reporter gene and introduced into pituitary and non-pituitary cell lines. A 30-fold increase in rPRL promoter activity was observed in GH4 rat pituitary tumor cells compared to nonpituitary Rat2 fibroblast and HeLa cervical carcinoma cells. About 45% of this cell-specific promoter activity was competed by a plasmid containing the -67 to -45 rPRL promoter region, which is the most proximal binding site for a lactotroph-specific factor. Compared to a -425 rPRL construct, transfection with rPRL 5'-end points of -212, -178, and -127 contained 23%, 45%, and 1%, respectively, of luciferase activity. Forskolin stimulation resulted in a 10-fold induction of all the rPRL promoter fragments tested. Of note, a -127 deletion which was devoid of any basal promoter activity was also induced 10-fold by forskolin. The forskolin effect was abolished when GH4 rat pituitary cells were cotransfected with a plasmid encoding a protein kinase A inhibitor, indicating protein kinase A is involved in the activation mechanism. These data document that both positive and negative effectors influence basal rPRL promoter activity. Furthermore, the minimum sequences required for pituitary-specific rPRL promoter activity are altered by intracellular cAMP levels. Taken together, the data indicate that hormone-activated and cell-specific factors may interact to establish a particular setpoint for rPRL gene expression.